Central Circadian Oscillator Research Articles

Effects of adenylates on the circadian interaction of KaiB with the KaiC complex in the reconstituted cyanobacterial Kai protein oscillator.

Cyanobacteria are photosynthetic prokaryotes that possess circadian oscillators. Clock proteins, KaiA, KaiB, KaiC compose the central circadian oscillator, which can be reconstituted in vitro in the presence of ATP. KaiC has ATPase, autokinase, and autophosphatase enzymatic activities. These activities are modulated by protein-protein interactions among the Kai proteins. The interaction of KaiB with the KaiC complex shows a circadian rhythm in the reconstituted system. We previously developed a quantitative, real-time monitoring system for the dynamic behavior of the complex using fluorescence correlation spectroscopy. Here, we examined the effects of ATP and ADP on the rhythmic interaction of KaiB. We show that increased concentration of ATP or ADP shortened period length. Adding ADP to the Kai protein oscillation shifted its phase in a phase-dependent manner. These results provide insight into how circadian oscillation entrainment mechanism is linked to cellular metabolism.

Suprachiasmatic nucleus function and circadian entrainment are modulated by G protein-coupled inwardly rectifying (GIRK) channels.

G protein signalling within the central circadian oscillator, the suprachiasmatic nucleus (SCN), is essential for conveying time-of-day information. We sought to determine whether G protein-coupled inwardly rectifying potassium channels (GIRKs) modulate SCN physiology and circadian behaviour. We show that GIRK current and GIRK2 protein expression are greater during the day. Pharmacological inhibition of GIRKs and genetic loss of GIRK2 depolarized the day-time resting membrane potential of SCN neurons compared to controls. Behaviourally, GIRK2 knockout (KO) mice failed to shorten free running period in response to wheel access in constant darkness and entrained more rapidly to a 6 h advance of a 12 h:12 h light-dark (LD) cycle than wild-type (WT) littermate controls. We next examined whether these effects were due to disrupted signalling of neuropeptide Y (NPY), which is known to mediate non-photic phase shifts, attenuate photic phase shifts and activate GIRKs. Indeed, GIRK2 KO SCN slices had significantly fewer silent cells in response to NPY, likely contributing to the absence of NPY-induced phase advances of PER2::LUC rhythms in organotypic SCN cultures from GIRK2 KO mice. Finally, GIRK channel activation is sufficient to cause a non-photic-like phase advance of PER2::LUC rhythms on a Per2(Luc+/-) background. These results suggest that rhythmic regulation of GIRK2 protein and channel function in the SCN contributes to day-time resting membrane potential, providing a mechanism for the fine tuning responses to non-photic and photic stimuli. Further investigation could provide insight into disorders with circadian disruption comorbidities such as epilepsy and addiction, in which GIRK channels have been implicated.

Melatonin, noncoding RNAs, messenger RNA stability and epigenetics--evidence, hints, gaps and perspectives.

Melatonin is a highly pleiotropic regulator molecule, which influences numerous functions in almost every organ and, thus, up- or down-regulates many genes, frequently in a circadian manner. Our understanding of the mechanisms controlling gene expression is actually now expanding to a previously unforeseen extent. In addition to classic actions of transcription factors, gene expression is induced, suppressed or modulated by a number of RNAs and proteins, such as miRNAs, lncRNAs, piRNAs, antisense transcripts, deadenylases, DNA methyltransferases, histone methylation complexes, histone demethylases, histone acetyltransferases and histone deacetylases. Direct or indirect evidence for involvement of melatonin in this network of players has originated in different fields, including studies on central and peripheral circadian oscillators, shift work, cancer, inflammation, oxidative stress, aging, energy expenditure/obesity, diabetes type 2, neuropsychiatric disorders, and neurogenesis. Some of the novel modulators have also been shown to participate in the control of melatonin biosynthesis and melatonin receptor expression. Future work will need to augment the body of evidence on direct epigenetic actions of melatonin and to systematically investigate its role within the network of oscillating epigenetic factors. Moreover, it will be necessary to discriminate between effects observed under conditions of well-operating and deregulated circadian clocks, and to explore the possibilities of correcting epigenetic malprogramming by melatonin.

Acute dim light at night increases body mass, alters metabolism, and shifts core body temperature circadian rhythms

The circadian system is primarily entrained by the ambient light environment and is fundamentally linked to metabolism. Mounting evidence suggests a causal relationship among aberrant light exposure, shift work, and metabolic disease. Previous research has demonstrated deleterious metabolic phenotypes elicited by chronic (>4 weeks) exposure to dim light at night (DLAN) (∼5 lux). However, the metabolic effects of short-term (<2 weeks) exposure to DLAN are unspecified. We hypothesized that metabolic alterations would arise in response to just 2 weeks of DLAN. Specifically, we predicted that mice exposed to dim light would gain more body mass, alter whole body metabolism, and display altered body temperature (Tb) and activity rhythms compared to mice maintained in dark nights. Our data largely support these predictions; DLAN mice gained significantly more mass, reduced whole body energy expenditure, increased carbohydrate over fat oxidation, and altered temperature circadian rhythms. Importantly, these alterations occurred despite similar activity locomotor levels (and rhythms) and total food intake between groups. Peripheral clocks are potently entrained by body temperature rhythms, and the deregulation of body temperature we observed may contribute to metabolic problems due to “internal desynchrony” between the central circadian oscillator and temperature sensitive peripheral clocks. We conclude that even relatively short-term exposure to low levels of nighttime light can influence metabolism to increase mass gain.

Wheels within wheels: the plant circadian system

Circadian clocks integrate environmental signals with internal cues to coordinate diverse physiological outputs so that they occur at the most appropriate season or time of day. Recent studies using systems approaches, primarily in Arabidopsis, have expanded our understanding of the molecular regulation of the central circadian oscillator and its connections to input and output pathways. Similar approaches have also begun to reveal the importance of the clock for key agricultural traits in crop species. In this review, we discuss recent developments in the field, including a new understanding of the molecular architecture underlying the plant clock; mechanistic links between clock components and input and output pathways; and our growing understanding of the importance of clock genes for agronomically important traits.

Current Perspectives in Uterine Research: Reviews From the Society for Gynecologic Investigation Myometrium and Parturition Satellite Symposia

It is difficult to imagine examples of greater physiological adaptations than those of the uteroplacental unit during pregnancy. The sheer scale of molecular, cellular and tissue remodeling, organogenesis, and organism development in a relatively short period of time seems the epitome of ‘‘systems biology.’’ The processes are challenging to comprehend, and investigate experimentally, but undoubtedly fascinating. Efforts to understand the physiology and pathology of these phenomena are of prime importance due to the considerable health and socioeconomic pressures brought to bear by the high incidences of compromised pregnancies and, especially, preterm birth (PTB). The World Health Organization-sponsored studies equate the worldwide incidence of PTB to be approximately 13 million births per year with approximately 1 million neonatal deaths attributable to PTB. If improved diagnosis and therapeutic treatment of PTB are to be achieved, an integrated understanding of the physiological basis of pregnancy and parturition is required. With this in mind, each year the Myometrium and Parturition Satellite Symposium of the Society for Gynecologic Investigation meeting gathers international experts in this field to share their experimental findings. We are delighted that several plenary speakers from the 2011 and 2012 symposia have provided reviews on their topics for this issue of Reproductive Sciences. The articles provide an up-to-date compendium that integrates many of the important facets of uteroplacental remodeling and function leading to term and preterm labor. Jerome Strauss (Virginia) introduces the importance of dynamic remodeling of the extracellular matrix (ECM) and its molecular components in uteroplacental tissues. With a focus on fetal membrane interfaces, he summarizes the ECMs’ multiplicity of roles including receptor signaling, cytokine/ growth factor sequestration, and tensile strength. Among the important molecular components are fetal fibronectin, hyaluronan, and up to 28 collagens, regulated by different metallomatrix proteinases. Mutations in many genes encoding such ECM proteins are associated with increased incidence of preterm premature rupture of membranes and the roles of environmental and/or epigenetic factors are being increasingly elucidated. In a similar vein, Shynlova et al (Toronto, Canada) describe pregnancy in terms of progressive changes of tissue remodeling under the control of localized inflammatory events (physiological inflammation). The authors posit a carefully structured theory whereby spatially restricted inflammatory events are under the influence of ever-changing endocrine (and a focus is given to the role of progesterone) and mechanical inputs activating a rich milieu of chemokine/cytokine signaling. These, in turn, serve to coordinate diverse processes such as decidualization, myometrial cell proliferation and growth, leukocyte migration, cervical remodeling, and postparturient involution. Olcese et al (Tallahassee, USA) review an oft-neglected consideration of the mechanisms responsible for the timing of parturition. They too give credence to the notion that pregnancy and parturition involve dynamic physiological adaptations—in this case that uterine contractions of labor may be initiated by activity of the central circadian oscillator of the maternal hypothalamic suprachiasmatic nuclei via the feedback ‘‘gating’’ actions of neuroendocrine outputs. Of key importance may well be circadian secretion of melatonin given recent interest in the uterotonic actions of this hormone and uterine expression of melatonin receptors. Regulation of contractile activity of the uterus is also the focus of reviews by Europe-Finner et al (Newcastle, UK) and Wim Lammers (Al Ain, UAE). The former concentrates on intracellular signaling and describes the possibility that posttranslational modification by acetylation may be a mechanism of wider significance to the uterus than first realized when considering ‘‘‘only’’ nuclear events of histone modification, chromatin remodeling, and gene transcription. They suggest

Circadian adaptations to meal timing: neuroendocrine mechanisms

Circadian rhythms of behavior and physiology are generated by central and peripheral circadian oscillators entrained by periodic environmental or physiological stimuli. A master circadian pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) is directly entrained by daily light-dark (LD) cycles, and coordinates the timing of other oscillators by direct and indirect neural, hormonal and behavioral outputs. The daily rhythm of food intake provides stimuli that entrain most peripheral and central oscillators, some of which can drive a daily rhythm of food anticipatory activity if food is restricted to one daily mealtime. The location of food-entrainable oscillators (FEOs) that drive food anticipatory rhythms, and the food-related stimuli that entrain these oscillators, remain to be clarified. Here, we critically examine the role of peripheral metabolic hormones as potential internal entrainment stimuli or outputs for FEOs controlling food anticipatory rhythms in rats and mice. Hormones for which data are available include corticosterone, ghrelin, leptin, insulin, glucagon, and glucagon-like peptide 1. All of these hormones exhibit daily rhythms of synthesis and secretion that are synchronized by meal timing. There is some evidence that ghrelin and leptin modulate the expression of food anticipatory rhythms, but none of the hormones examined so far are necessary for entrainment. Ghrelin and leptin likely modulate food-entrained rhythms by actions in hypothalamic circuits utilizing melanocortin and orexin signaling, although again food-entrained behavioral rhythms can persist in lesion and gene knockout models in which these systems are disabled. Actions of these hormones on circadian oscillators in central reward circuits remain to be evaluated. Food-entrained activity rhythms are likely mediated by a distributed system of circadian oscillators sensitive to multiple feeding related inputs. Metabolic hormones appear to play a modulatory role within this system.

SRF’ing Around the Clock

Central circadian clock oscillators, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, align peripheral clock systems with geosynchronous time. Gerber et al. (2013) identify actin polymerization and serum response factor (SRF) activation as key steps linking the central master clock to peripheral oscillators.

Melatonin and clock genes expression in the cardiovascular system

Generation of circadian oscillations is based on rhythmic expression of clock genes and subsequent post-transcriptional and post-translational modifications. In addition to the central circadian oscillator - the suprachiasmatic nucleus (SCN), peripheral oscillators have been demonstrated in many tissues, including the heart and blood vessels. Melatonin mediates cyclic lighting conditions to rhythmic endocrine signal and is able to synchronize neuronal firing in the SCN via membrane receptors. Clock gene expression is melatonin sensitive in the pars tuberalis, genes cry1 and tim1 respond to single injection while neurod1 and npas4 are influenced via long lasting mechanisms. In the rat heart, melatonin phase advanced expression of per2 and bmal1 independently from its effects on the SCN. Melatonin is an important endogenous signal able to synchronize circadian oscillations in the cardiovascular system. It may be effective especially in situations when the circadian control is weakened or organism must adapt to rapid changes in rhythmic environmental conditions.

Transcriptional corepressor TOPLESS complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription

Circadian clocks are ubiquitous molecular time-keeping mechanisms that coordinate physiology and metabolism and provide an adaptive advantage to higher plants. The central oscillator of the plant clock is composed of interlocked feedback loops that involve multiple repressive factors acting throughout the circadian cycle. Pseudo response regulators (PRRs) comprise a five-member family that is essential to the function of the central oscillator. PRR5, PRR7, and PRR9 can bind the promoters of the core clock genes circadian clock associated 1 (CCA1) and late elongated hypocotyl (LHY) to restrict their expression to near dawn, but the mechanism has been unclear. Here we report that members of the plant Groucho/Tup1 corepressor family, topless/topless-related (TPL/TPR), interact with these three PRR proteins at the CCA1 and LHY promoters to repress transcription and alter circadian period. This activity is diminished in the presence of the inhibitor trichostatin A, indicating the requirement of histone deacetylase for full TPL activity. Additionally, a complex of PRR9, TPL, and histone deacetylase 6, can form in vivo, implicating this tripartite association as a central repressor of circadian gene expression. Our findings show that the TPL/TPR corepressor family are components of the central circadian oscillator mechanism and reinforces the role of this family as central to multiple signaling pathways in higher plants.

Aging Differentially Affects the Re-entrainment Response of Central and Peripheral Circadian Oscillators

Aging produces a decline in the amplitude and precision of 24 h behavioral, endocrine, and metabolic rhythms, which are regulated in mammals by a central circadian pacemaker within the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. Disruption of the circadian system, as experienced during transmeridian travel, can lead to adverse health consequences, particularly in the elderly. To test the hypothesis that age-related changes in the response to simulated jet lag will reflect altered circadian function, we examined re-entrainment of central and peripheral oscillators from young and old PER2::luciferase mice. As in previous studies, locomotor activity rhythms in older mice required more days to re-entrain following a shift than younger mice. At the tissue level, effects of age on baseline entrainment were evident, with older mice displaying earlier phases for the majority of peripheral oscillators studied and later phases for cells within most SCN subregions. Following a 6 h advance of the light:dark cycle, old mice displayed slower rates of re-entrainment for peripheral tissues but a larger, more rapid SCN response compared to younger mice. Thus, aging alters the circadian timing system in a manner that differentially affects the re-entrainment responses of central and peripheral circadian clocks. This pattern of results suggests that a major consequence of aging is a decrease in pacemaker amplitude, which would slow re-entrainment of peripheral oscillators and reduce SCN resistance to external perturbation.

Circadian changes in long noncoding RNAs in the pineal gland

Long noncoding RNAs (lncRNAs) play a broad range of biological roles, including regulation of expression of genes and chromosomes. Here, we present evidence that lncRNAs are involved in vertebrate circadian biology. Differential night/day expression of 112 lncRNAs (0.3 to >50 kb) occurs in the rat pineal gland, which is the source of melatonin, the hormone of the night. Approximately one-half of these changes reflect nocturnal increases. Studies of eight lncRNAs with 2- to >100-fold daily rhythms indicate that, in most cases, the change results from neural stimulation from the central circadian oscillator in the suprachiasmatic nucleus (doubling time = 0.5-1.3 h). Light exposure at night rapidly reverses (halving time = 9-32 min) levels of some of these lncRNAs. Organ culture studies indicate that expression of these lncRNAs is regulated by norepinephrine acting through cAMP. These findings point to a dynamic role of lncRNAs in the circadian system.

Brain-Specific Rescue of Clock Reveals System-Driven Transcriptional Rhythms in Peripheral Tissue

The circadian regulatory network is organized in a hierarchical fashion, with a central oscillator in the suprachiasmatic nuclei (SCN) orchestrating circadian oscillations in peripheral tissues. The nature of the relationship between central and peripheral oscillators, however, is poorly understood. We used the tetOFF expression system to specifically restore Clock function in the brains of ClockΔ19 mice, which have compromised circadian clocks. Rescued mice showed normal locomotor rhythms in constant darkness, with activity period lengths approximating wildtype controls. We used microarray analysis to assess whether brain-specific rescue of circadian rhythmicity was sufficient to restore circadian transcriptional output in the liver. Compared to Clock mutants, Clock-rescue mice showed significantly larger numbers of cycling transcripts with appropriate phase and period lengths, including many components of the core circadian oscillator. This indicates that the SCN oscillator overcomes local circadian defects and signals directly to the molecular clock. Interestingly, the vast majority of core clock genes in liver were responsive to Clock expression in the SCN, suggesting that core clock genes in peripheral tissues are intrinsically sensitive to SCN cues. Nevertheless, most circadian output in the liver was absent or severely low-amplitude in Clock-rescue animals, demonstrating that the majority of peripheral transcriptional rhythms depend on a fully functional local circadian oscillator. We identified several new system-driven rhythmic genes in the liver, including Alas1 and Mfsd2. Finally, we show that 12-hour transcriptional rhythms (i.e., circadian “harmonics") are disrupted by Clock loss-of-function. Brain-specific rescue of Clock converted 12-hour rhythms into 24-hour rhythms, suggesting that signaling via the central circadian oscillator is required to generate one of the two daily peaks of expression. Based on these data, we conclude that 12-hour rhythms are driven by interactions between central and peripheral circadian oscillators.

Simultaneous effect of melatonin on supraoesophageal ganglion spontaneous electrical activity and photoreceptor electroretinogram amplitude in crayfish

In crayfish, the supraoesophageal ganglion and the retina are neuronal structures whose electrical activities follow a circadian rhythm. The hormone melatonin (MEL) is an indoleamine, conserved from unicellular organisms to mammals, that is released rhythmically with a peak occurring in the darkness period. In vertebrates, MEL acts on numerous central and peripheral structures and participates in the modulation of circadian rhythms. The main purpose of this study was to present evidence of the role of MEL in crayfish circadian physiology, simultaneously recording the electrical activity of both the supraoesophageal ganglion and the retina under the free-running condition. We found that at circadian time (CT) 2, when the concentration of endogenous MEL is low, injection of this indoleamine into intact animals elicited a decrease in the level of spontaneous electrical activity recorded from the supraoesophageal ganglion and enhancement of electroretinogram amplitude. The MEL-induced effect mimics the level of these activities that was observed when the endogenous MEL concentration is high. These results suggest that MEL acts, simultaneously on both central and peripheral circadian oscillators, as a mediator to maintain the crayfish circadian system organization under the free-running condition.

Unproductive alternative splicing and nonsense mRNAs: A widespread phenomenon among plant circadian clock genes

BackgroundRecent mapping of eukaryotic transcriptomes and spliceomes using massively parallel RNA sequencing (RNA-seq) has revealed that the extent of alternative splicing has been considerably underestimated. Evidence also suggests that many pre-mRNAs undergo unproductive alternative splicing resulting in incorporation of in-frame premature termination codons (PTCs). The destinies and potential functions of the PTC-harboring mRNAs remain poorly understood. Unproductive alternative splicing in circadian clock genes presents a special case study because the daily oscillations of protein expression levels require rapid and steep adjustments in mRNA levels.ResultsWe conducted a systematic survey of alternative splicing of plant circadian clock genes using RNA-seq and found that many Arabidopsis thaliana circadian clock-associated genes are alternatively spliced. Results were confirmed using reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and/or Sanger sequencing. Intron retention events were frequently observed in mRNAs of the CCA1/LHY-like subfamily of MYB transcription factors. In contrast, the REVEILLE2 (RVE2) transcript was alternatively spliced via inclusion of a "poison cassette exon" (PCE). The PCE type events introducing in-frame PTCs are conserved in some mammalian and plant serine/arginine-rich splicing factors. For some circadian genes such as CCA1 the ratio of the productive isoform (i.e., a representative splice variant encoding the full-length protein) to its PTC counterpart shifted sharply under specific environmental stress conditions.ConclusionsOur results demonstrate that unproductive alternative splicing is a widespread phenomenon among plant circadian clock genes that frequently generates mRNA isoforms harboring in-frame PTCs. Because LHY and CCA1 are core components of the plant central circadian oscillator, the conservation of alternatively spliced variants between CCA1 and LHY and for CCA1 across phyla [2] indicates a potential role of nonsense transcripts in regulation of circadian rhythms. Most of the alternatively spliced isoforms harbor in-frame PTCs that arise from full or partial intron retention events. However, a PTC in the RVE2 transcript is introduced through a PCE event. The conservation of AS events and modulation of the relative abundance of nonsense isoforms by environmental and diurnal conditions suggests possible regulatory roles for these alternatively spliced transcripts in circadian clock function. The temperature-dependent expression of the PTC transcripts among members of CCA1/LHY subfamily indicates that alternative splicing may be involved in regulation of the clock temperature compensation mechanism.ReviewersThis article was reviewed by Dr. Eugene Koonin, Dr. Chungoo Park (nominated by Dr. Kateryna Makova), and Dr. Marcelo Yanovsky (nominated by Dr. Valerian Dolja).

Coordinated transcriptional regulation underlying the circadian clock in Arabidopsis

The circadian clock controls many metabolic, developmental and physiological processes in a time-of-day-specific manner in both plants and animals. The photoreceptors involved in the perception of light and entrainment of the circadian clock have been well characterized in plants. However, how light signals are transduced from the photoreceptors to the central circadian oscillator, and how the rhythmic expression pattern of a clock gene is generated and maintained by diurnal light signals remain unclear. Here, we show that in Arabidopsis thaliana, FHY3, FAR1 and HY5, three positive regulators of the phytochrome A signalling pathway, directly bind to the promoter of ELF4, a proposed component of the central oscillator, and activate its expression during the day, whereas the circadian-controlled CCA1 and LHY proteins directly suppress ELF4 expression periodically at dawn through physical interactions with these transcription-promoting factors. Our findings provide evidence that a set of light- and circadian-regulated transcription factors act directly and coordinately at the ELF4 promoter to regulate its cyclic expression, and establish a potential molecular link connecting the environmental light-dark cycle to the central oscillator.

Circadian rhythms and cognition in schizophrenia

Cognitive impairment is a core feature of schizophrenia. Evidence in this Journal suggests that disrupted circadian synchronisation of sleep-wake phases may be one cause. Circadian synchronisation depends on the external input of light-dark cues and the integrity of central nervous system circadian oscillators; these may represent new interventional targets to improve cognition.

Mammalian Molecular Clocks

As a consequence of the Earth's rotation, almost all organisms experience day and night cycles within a 24-hr period. To adapt and synchronize biological rhythms to external daily cycles, organisms have evolved an internal time-keeping system. In mammals, the master circadian pacemaker residing in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus generates circadian rhythmicity and orchestrates numerous subsidiary local clocks in other regions of the brain and peripheral tissues. Regardless of their locations, these circadian clocks are cell-autonomous and self-sustainable, implicating rhythmic oscillations in a variety of biochemical and metabolic processes. A group of core clock genes provides interlocking molecular feedback loops that drive the circadian rhythm even at the single-cell level. In addition to the core transcription/translation feedback loops, post-translational modifications also contribute to the fine regulation of molecular circadian clocks. In this article, we briefly review the molecular mechanisms and post-translational modifications of mammalian circadian clock regulation. We also discuss the organization of and communication between central and peripheral circadian oscillators of the mammalian circadian clock.

Identification of two Amino Acids in the C-terminal Domain of Mouse CRY2 Essential for PER2 Interaction

BackgroundCryptochromes (CRYs) are a class of flavoprotein blue-light signaling receptors found in plants and animals, and they control plant development and the entrainment of circadian rhythms. They also act as integral parts of the central circadian oscillator in humans and other animals. In mammals, the CLOCK-BMAL1 heterodimer activates transcription of the Per and Cry genes as well as clock-regulated genes. The PER2 proteins interact with CRY and CKIε, and the resulting ternary complexes translocate into the nucleus, where they negatively regulate the transcription of Per and Cry core clock genes and other clock-regulated output genes. Recent studies have indicated that the extended C-termini of the mammalian CRYs, as compared to photolyase proteins, interact with PER proteins.ResultsWe identified a region on mCRY2 (between residues 493 and 512) responsible for direct physical interaction with mPER2 by mammalian two-hybrid and co-immunoprecipitation assays. Moreover, using oligonucleotide-based degenerate PCR, we discovered that mutation of Arg-501 and Lys-503 of mCRY2 within this C-terminal region totally abolishes interaction with PER2.ConclusionsOur results identify mCRY2 amino acid residues that interact with the mPER2 binding region and suggest the potential for rational drug design to inhibit CRYs for specific therapeutic approaches.

Circadian Input Kinases and Their Homologs in Cyanobacteria: Evolutionary Constraints Versus Architectural Diversification

The circadian input kinase A (cikA) gene encodes a protein relaying environmental signal to the central circadian oscillator in cyanobacteria. The CikA protein has a variable architecture and usually consists of four tandemly arrayed domains: GAF, histidine kinase (HisKA), histidine kinase-like ATPase (HATPase_c), and a pseudo-receiver (REC). Among them, HisKA and HATPase_c are the least polymorphic, and REC is not present in heterocystic filamentous cyanobacteria. CikA contains several conserved motifs that are likely important for circadian function. There are at least three types of circadian systems, each of which possesses a different set of circadian genes. The originally described circadian system (kaiABC system) possesses both cikA and kaiA, while the others lack either only cikA (kaiABC (Delta)) or both (kaiBC). The results we obtained allowed us to approximate the time of the cikA origin to be about 2600-2200 MYA and the time of its loss in the species with the kaiABC (Delta) or kaiBC system between 1100 and 600 MYA. Circadian specialization of CikA, as opposed to its non-circadian homologs, is a result of several factors, including the unique conserved domain architecture and high evolutionary constraints of some domains and regions, which were previously identified as critical for the circadian function of the gene.