Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Impact of circadian clock protein Bmal1 on experimentally-induced periodontitis-associated renal injury.

OBJECTIVES: To investigate the mechanism of circadian clock protein Bmal1 (Bmal1) on renal injury with chronic periodontitis, we established an experimental rat periodontitis model. METHODS: Twelve male Wistar rats were randomly divided into control and periodontitis groups (n=6, each group). The first maxillary molars on both sides of the upper jaw of rats with periodontitis were ligated by using orthodontic ligature wires, whereas the control group received no intervention measures. After 8 weeks, clinical periodontal parameters, including probing depth, bleeding index, and tooth mobility, were evaluated in both groups. Micro-CT scanning and three-dimensional image reconstruction were performed on the maxillary bones of the rats for the assessment of alveolar bone resorption. Histopatholo-gical observations of periodontal and renal tissues were conducted using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. Renal function indicators, such as creatinine, albumin, and blood urea nitrogen levels, and oxidative stress markers, including superoxide dismutase, glutathione, and malondialdehyde levels, were measured using biochemical assay kits. MitoSOX red staining was used to detect reactive oxygen species (ROS) content in the kidneys. The gene and protein expression levels of Bmal1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in rat renal tissues were assessed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. RESULTS: Micro-CT and HE staining results showed significant bone resorption and attachment loss in the maxillary first molar region of the periodontitis group. Histological examination through HE and PAS staining revealed substantial histopathological damage to the renal tissues of the rats in the periodontitis group. The findings of the assessment of renal function and oxidative stress markers indicated that the periodontitis group exhibited abnormal levels of oxidative stress, whereas the renal function levels showed abnormalities without statistical significance. MitoSOX Red staining results showed that the content of ROS in the renal tissue of the periodontitis group was significantly higher than that of the control group, and RT-qPCR and immunohistochemistry results showed that the expression levels of Bmal1, Nrf2, and HO-1 in the renal tissues of the rats in the periodontitis group showed a decreasing trend. CONCLUSIONS: Circadian clock protein Bmal1 plays an important role in the oxidative damage process involved in the renal of rats with periodontitis.

Dynamic encoding of temperature in the central circadian circuit coordinates physiological activities.

The circadian clock regulates animal physiological activities. How temperature reorganizes circadian-dependent physiological activities remains elusive. Here, using in-vivo two-photon imaging with the temperature control device, we investigated the response of the Drosophila central circadian circuit to temperature variation and identified that DN1as serves as the most sensitive temperature-sensing neurons. The circadian clock gate DN1a's diurnal temperature response. Trans-synaptic tracing, connectome analysis, and functional imaging data reveal that DN1as bidirectionally targets two circadian neuronal subsets: activity-related E cells and sleep-promoting DN3s. Specifically, behavioral data demonstrate that the DN1a-E cell circuit modulates the evening locomotion peak in response to cold temperature, while the DN1a-DN3 circuit controls the warm temperature-induced nocturnal sleep reduction. Our findings systematically and comprehensively illustrate how the central circadian circuit dynamically integrates temperature and light signals to effectively coordinate wakefulness and sleep at different times of the day, shedding light on the conserved neural mechanisms underlying temperature-regulated circadian physiology in animals.

Circadian disturbances by altering the light-dark cycle negatively affects hematopoietic function of bone marrow in mice.

Circadian rhythms in metabolically active tissues are crucial for maintaining physical health. Circadian disturbance (CD) can cause various health issues, such as metabolic abnormalities and immune and cognitive dysfunctions. However, studies on the role of CD in immune cell development and differentiation, as well as the rhythmic expression of the core clock genes and their altered expression under CD, remain unclear. Therefore, we exposed C57bl/6j mice to repeated reversed light-dark cycles for 90 days to research the effects of CD on bone marrow (BM) hematopoietic function. We also researched the effects of CD on endogenous circadian rhythms, temporally dependent expression in peripheral blood and myeloid leukocytes, environmental homeostasis within BM, and circadian oscillations of hematopoietic-extrinsic cues. Our results confirmed that when the light and dark cycles around mice were frequently reversed, the circadian rhythmic expression of the two main circadian rhythm markers, the hypothalamic clock gene, and serum melatonin, was disturbed, indicating that the body was in a state of endogenous CD. Furthermore, CD altered the temporally dependent expression of peripheral blood and BM leukocytes and destroyed environmental homeostasis within the BM as well as circadian oscillations of hematopoietic-extrinsic cues, which may negatively affect BM hematopoiesis in mice. Collectively, these results demonstrate that circadian rhythms are vital for maintaining health and suggest that the association between CD and hematopoietic dysfunction warrants further investigation.

Molecular circadian rhythms are robust in marine annelids lacking rhythmic behavior.

The circadian clock controls behavior and metabolism in various organisms. However, the exact timing and strength of rhythmic phenotypes can vary significantly between individuals of the same species. This is highly relevant for rhythmically complex marine environments where organismal rhythmic diversity likely permits the occupation of different microenvironments. When investigating circadian locomotor behavior of Platynereis dumerilii, a model system for marine molecular chronobiology, we found strain-specific, high variability between individual worms. The individual patterns were maintained for several weeks. A diel head transcriptome comparison of behaviorally rhythmic versus arrhythmic wild-type worms showed that 24-h cycling of core circadian clock transcripts is identical between both behavioral phenotypes. While behaviorally arrhythmic worms showed a similar total number of cycling transcripts compared to their behaviorally rhythmic counterparts, the annotation categories of their transcripts, however, differed substantially. Consistent with their locomotor phenotype, behaviorally rhythmic worms exhibit an enrichment of cycling transcripts related to neuronal/behavioral processes. In contrast, behaviorally arrhythmic worms showed significantly increased diel cycling for metabolism- and physiology-related transcripts. The prominent role of the neuropeptide pigment-dispersing factor (PDF) in Drosophila circadian behavior prompted us to test for a possible functional involvement of Platynereis pdf. Differing from its role in Drosophila, loss of pdf impacts overall activity levels but shows only indirect effects on rhythmicity. Our results show that individuals arrhythmic in a given process can show increased rhythmicity in others. Across the Platynereis population, rhythmic phenotypes exist as a continuum, with no distinct "boundaries" between rhythmicity and arrhythmicity. We suggest that such diel rhythm breadth is an important biodiversity resource enabling the species to quickly adapt to heterogeneous or changing marine environments. In times of massive sequencing, our work also emphasizes the importance of time series and functional tests.

Meal timing and its role in obesity and associated diseases.

Meal timing emerges as a crucial factor influencing metabolic health that can be explained by the tight interaction between the endogenous circadian clock and metabolic homeostasis. Mistimed food intake, such as delayed or nighttime consumption, leads to desynchronization of the internal circadian clock and is associated with an increased risk for obesity and associated metabolic disturbances such as type 2 diabetes and cardiovascular diseases. Conversely, meal timing aligned with cellular rhythms can optimize the performance of tissues and organs. In this review, we provide an overview of the metabolic effects of meal timing and discuss the underlying mechanisms. Additionally, we explore factors influencing meal timing, including internal determinants such as chronotype and genetics, as well as external influences like social factors, cultural aspects, and work schedules. This review could contribute to defining meal-timing-based recommendations for public health initiatives and developing guidelines for effective lifestyle modifications targeting the prevention and treatment of obesity and associated metabolic diseases. Furthermore, it sheds light on crucial factors that must be considered in the design of future food timing intervention trials.

Receptor-Mediated and Receptor-Independent Actions of Melatonin in Vertebrates.

Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine that is synthesized from tryptophan in the pineal glands of vertebrates through four enzymatic reactions. Melatonin is a quite unique bioactive substance, characterized by a combination of both receptor-mediated and receptor-independent actions, which promote the diverse effects of melatonin. One of the main functions of melatonin, via its membrane receptors, is to regulate the circadian or seasonal rhythm. In mammals, light information, which controls melatonin synthesis, is received in the eye, and transmitted to the pineal gland, via the suprachiasmatic nucleus, where the central clock is located. Alternatively, in many vertebrates other than mammals, the pineal gland cells, which are involved in melatonin synthesis and secretion and in the circadian clock, directly receive light. Recently, it has been reported that melatonin possesses several metabolic functions, which involve bone and glucose, in addition to regulating the circadian rhythm. Melatonin improves bone strength by inhibiting osteoclast activity. It is also known to maintain brain activity during sleep by increasing glucose uptake at night, in an insulin-independent manner. Moreover, as a non-receptor-mediated action, melatonin has antioxidant properties. Melatonin has been proven to be a potent free radical scavenger and a broad-spectrum antioxidant, even protecting organisms against radiation from space. Melatonin is a ubiquitously distributed molecule and is found in bacteria, unicellular organisms, fungi, and plants. It is hypothesized that melatonin initially functioned as an antioxidant, then, in vertebrates, it combined this role with the ability to regulate rhythm and metabolism, via its receptors.

A Luciferase Imaging-Based Assay for Studying Temperature Compensation of the Circadian Clock.

The pace of circadian rhythms remains relatively unchanged across a physiologically relevant range of temperatures, a phenomenon known as temperature compensation. Temperature compensation is a defining characteristic of circadian rhythms, ensuring that clock-regulated processes occur at approximately the same time of day across a wide range of conditions. Despite the identification of several genes involved in the regulation of temperature compensation, the molecular mechanisms underlying this process are still not well understood. High-throughput assays of circadian period are essential for the investigation of temperature compensation. In this chapter, we present a luciferase imaging-based method that enables robust and accurate examination of temperature compensation in the plant circadian clock.

Dissection of Daytime and Nighttime Thermoresponsive Hypocotyl Elongation in Arabidopsis.

Hypocotyl elongation in Arabidopsis is widely utilized as a readout for phytochrome B (phyB) signaling and thermomorphogenesis. Hypocotyl elongation is gated by the circadian clock and, therefore, it occurs at distinct times depending on day length or seasonal cues. In short-day conditions, hypocotyl elongation occurs mainly at the end of nighttime when phyB reverts to the inactive form. In contrast, in long-day conditions, hypocotyl elongation occurs during the daytime when phyB is in the photoactivated form. Warm temperatures can induce hypocotyl growth in both long-day and short-day conditions. However, the corresponding daytime and nighttime temperature responses reflect distinct underpinning mechanisms. Here, we describe assays for dissecting the mechanisms between daytime and nighttime thermoresponsive hypocotyl elongation.

Analysis of Phase Separation of EARLY FLOWERING 3.

Phase separation is an important mechanism for regulating various cellular functions. The EARLY FLOWERING 3 (ELF3) protein, an essential element of the EVENING COMPLEX (EC) involved in circadian clock regulation, has been shown to undergo phase separation. ELF3 is known to significantly influence elongation growth and flowering time regulation, and this is postulated to be due to whether the protein is in the dilute or phase-separated state. Here, we present a brief overview of methods for analyzing ELF3 phase separation in vitro, including the generation of phase diagrams as a function of pH and salt versus protein concentrations, optical microscopy, fluorescence recovery after photobleaching (FRAP), and turbidity assays.

Investigating Circadian Gating of Temperature Responsive Genes.

Understanding gene expression dynamics in the context of the time of day and temperature response is an important part of understanding plant thermotolerance in a changing climate. Performing "gating" experiments under constant conditions and light-dark cycles allows users to identify and dissect the contribution of the time of day and circadian clock to the dynamic nature of stress-responsive genes. Here, we describe the design of specific laboratory experiments in plants (Arabidopsis thaliana and bread wheat, Triticum aestivum) to investigate temporal responses to heat (1 h at 37 °C) or cold (3 h at 4 °C), and we include known marker genes that have circadian-gated responses to temperature changes.

The Trissin/TrissinR signaling pathway in the circadian network regulates evening activity in Drosophila melanogaster under constant dark conditions.

The circadian clock in Drosophila is governed by a neural network comprising approximately 150 neurons, known as clock neurons, which are intricately interconnected by various neurotransmitters. The neuropeptides that play functional roles in these clock neurons have been identified; however, the roles of some neuropeptides, such as Trissin, remain unclear. Trissin is expressed in lateral dorsal clock neurons (LNds), while its receptor, TrissinR, is expressed in dorsal neuron 1 (DN1) and LNds. In this study, we investigated the role of the Trissin/TrissinR signaling pathway within the circadian network in Drosophila melanogaster. Analysis involving our newly generated antibody against the Trissin precursor revealed that Trissin expression in the LNds cycles in a circadian manner. Behavioral analysis further demonstrated that flies with Trissin or TrissinR knockout or knockdown showed delayed evening activity offset under constant darkness conditions. Notably, this observed delay in evening activity offset in TrissinRNAi flies was restored via the additional knockdown of Ion transport peptide (ITP), indicating that the Trissin/TrissinR signaling pathway transmits information via ITP. Therefore, this pathway may be a key regulator of the timing of evening activity offset termination, orchestrating its effects in collaboration with the neuropeptide, ITP.

Temperature-Dependent Upregulation of Per2 Protein Expression Is Mediated by eIF2α Kinases PERK and PKR through PI3K Activation.

Temperature-dependent translational control of the core clock gene Per2 plays an important role in establishing entrainment of the circadian clock to physiological body temperature cycles. Previously, we found an involvement of the phosphatidylinositol 3-kinase (PI3K) in causing Per2 protein expression in response to a warm temperature shift (WTS) within a physiological range (from 35 to 38.5 °C). However, signaling pathway mediating the Per2 protein expression in response to WTS is only sparsely understood. Additional factor(s) other than PI3K remains unknown. Here we report the identification of eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), as a novel mediator of WTS-dependent Per2 protein expression. Canonically, eIF2α has been regarded as a major downstream target of PERK and PKR. However, we found that PERK and PKR mediate WTS response of Per2 in a manner not involving eIF2α. We observed that PERK and PKR serve as an upstream regulator of PI3K rather than eIF2α in the context of WTS-dependent Per2 protein expression. There have been studies reporting PI3K activation occurring depending on PERK and PKR, while its physiological contribution has remained elusive. Our finding therefore not only helps to enrich the knowledge of how WTS affects Per2 protein expression but also extends the region of cellular biology involving the PERK/PKR-mediated PI3K activation to include entrainment-mechanism of the circadian clock.

Diel Cycle Proteomics: Illuminating Molecular Dynamics in Purple Bacteria for Optimized Biotechnological Applications.

Circadian rhythms, characterized by approximately 24 h cycles, play a pivotal role in enabling various organisms to synchronize their biological activities with daily variations. While ubiquitous in Eukaryotes, circadian clocks remain exclusively characterized in Cyanobacteria among Prokaryotes. These rhythms are regulated by a core oscillator, which is controlled by a cluster of three genes: kaiA, kaiB, and kaiC. Interestingly, recent studies revealed rhythmic activities, potentially tied to a circadian clock, in other Prokaryotes, including purple bacteria such as Rhodospirillum rubrum, known for its applications in fuel and plastic bioproduction. However, the pivotal question of how light and dark cycles influence protein dynamics and the expression of putative circadian clock genes remains unexplored in purple non-sulfur bacteria. Unraveling the regulation of these molecular clocks holds the key to unlocking optimal conditions for harnessing the biotechnological potential of R. rubrum. Understanding how its proteome responds to different light regimes-whether under continuous light or alternating light and dark cycles-could pave the way for precisely fine-tuning bioproduction processes. Here, we report for the first time the expressed proteome of R. rubrum grown under continuous light versus light and dark cycle conditions using a shotgun proteomic analysis. In addition, we measured the impact of light regimes on the expression of four putative circadian clock genes (kaiB1, kaiB2, kaiC1, kaiC2) at the transcriptional and translational levels using RT-qPCR and targeted proteomic (MRM-MS), respectively. The data revealed significant effects of light conditions on the overall differential regulation of the proteome, particularly during the early growth stages. Notably, several proteins were found to be differentially regulated during the light or dark period, thus impacting crucial biological processes such as energy conversion pathways and the general stress response. Furthermore, our study unveiled distinct regulation of the four kai genes at both the mRNA and protein levels in response to varying light conditions. Deciphering the impact of the diel cycle on purple bacteria not only enhances our understanding of their ecology but also holds promise for optimizing their applications in biotechnology, providing valuable insights into the origin and evolution of prokaryotic clock mechanisms.

Circadian Biology and the Neurovascular Unit.

Mammalian physiology and cellular function are subject to significant oscillations over the course of every 24-hour day. It is likely that these daily rhythms will affect function as well as mechanisms of disease in the central nervous system. In this review, we attempt to survey and synthesize emerging studies that investigate how circadian biology may influence the neurovascular unit. We examine how circadian clocks may operate in neural, glial, and vascular compartments, review how circadian mechanisms regulate cell-cell signaling, assess interactions with aging and vascular comorbidities, and finally ask whether and how circadian effects and disruptions in rhythms may influence the risk and progression of pathophysiology in cerebrovascular disease. Overcoming identified challenges and leveraging opportunities for future research might support the development of novel circadian-based treatments for stroke.

Circadian Effects on Vascular Immunopathologies.

Circadian rhythms exert a profound impact on most aspects of mammalian physiology, including the immune and cardiovascular systems. Leukocytes engage in time-of-day-dependent interactions with the vasculature, facilitating the emigration to and the immune surveillance of tissues. This review provides an overview of circadian control of immune-vascular interactions in both the steady state and cardiovascular diseases such as atherosclerosis and infarction. Circadian rhythms impact both the immune and vascular facets of these interactions, primarily through the regulation of chemoattractant and adhesion molecules on immune and endothelial cells. Misaligned light conditions disrupt this rhythm, generally exacerbating atherosclerosis and infarction. In cardiovascular diseases, distinct circadian clock genes, while functioning as part of an integrated circadian system, can have proinflammatory or anti-inflammatory effects on these immune-vascular interactions. Here, we discuss the mechanisms and relevance of circadian rhythms in vascular immunopathologies.

Revisiting the role and mechanism of ELF3 in circadian clock modulation.

The gene encoding EARLY FLOWERING3 (ELF3) is necessary for photoperiodic flowering and the normal regulation of circadian rhythms. It provides important information at the cellular level to uncover the biological mechanisms that improve plant growth and development. ELF3 interactions with transcription factors such as BROTHER OF LUX ARRHYTHMO (BOA), LIGHT-REGULATED WD1 (LWD1), PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), PHYTOCHROME-INTERACTING FACTOR 7 (PIF7), and LUX ARRHYTHMO (LUX) suggest a role in evening complex (EC) independent pathways, demanding further investigation to elucidate the EC-dependent versus EC-independent mechanisms. The ELF3 regulation of flowering time about photoperiod and temperature variations can also optimize crop cultivation across diverse latitudes. In this review paper, we summarize how ELF3's role in the circadian clock and light-responsive flowering control in crops offers substantial potential for scientific advancement and practical applications in biotechnology and agriculture. Despite its essential role in crop adaptation, very little is known in many important crops. Consequently, comprehensive and targeted research is essential for extrapolating ELF3-related insights from Arabidopsis to other crops, utilizing both computational and experimental methodologies. This research should prioritize investigations into ELF3's protein-protein interactions, post-translational modifications, and genomic targets to elucidate its contribution to accurate circadian clock regulation.

Hypoxic Conditions Modulate Chondrogenesis through the Circadian Clock: The Role of Hypoxia-Inducible Factor-1α.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer transcription factor composed of an alpha and a beta subunit. HIF-1α is a master regulator of cellular response to hypoxia by activating the transcription of genes that facilitate metabolic adaptation to hypoxia. Since chondrocytes in mature articular cartilage reside in a hypoxic environment, HIF-1α plays an important role in chondrogenesis and in the physiological lifecycle of articular cartilage. Accumulating evidence suggests interactions between the HIF pathways and the circadian clock. The circadian clock is an emerging regulator in both developing and mature chondrocytes. However, how circadian rhythm is established during the early steps of cartilage formation and through what signaling pathways it promotes the healthy chondrocyte phenotype is still not entirely known. This narrative review aims to deliver a concise analysis of the existing understanding of the dynamic interplay between HIF-1α and the molecular clock in chondrocytes, in states of both health and disease, while also incorporating creative interpretations. We explore diverse hypotheses regarding the intricate interactions among these pathways and propose relevant therapeutic strategies for cartilage disorders such as osteoarthritis.

YTHDF1-CLOCK axis contributes to pathogenesis of allergic airway inflammation through LLPS.

N6-methyladenosine (m6A) modification has been implicated in many cell processes and diseases. YTHDF1, a translation-facilitating m6A reader, has not been previously shown to be related to allergic airway inflammation. Here, we report that YTHDF1 is highly expressed in allergic airway epithelial cells and asthmatic patients and that it influences proinflammatory responses. CLOCK, a subunit of the circadian clock pathway, is the direct target of YTHDF1. YTHDF1 augments CLOCK translation in an m6A-dependent manner. Allergens enhance the liquid-liquid phase separation (LLPS) of YTHDF1 and drive the formation of a complex comprising dimeric YTHDF1 and CLOCK mRNA, which is distributed to stress granules. Moreover, YTHDF1 strongly activates NLRP3 inflammasome production and interleukin-1ß secretion leading to airway inflammatory responses, but these phenotypes are abolished by deleting CLOCK. These findings demonstrate that YTHDF1 is an important regulator of asthmatic airway inflammation, suggesting a potential therapeutic target for allergic airway inflammation.